Neuritogenic peptides

ABSTRACT

The present invention relates to peptide compounds that are capable of stimulating neuronal differentiation, neurite outgrowth and survival of neural cells, and enhancing synaptic plasticity, learning and memory, methods of treating diseases and conditions of nervous system by administration of compositions comprising said compounds. The compounds and compositions of the invention include peptide sequences that are derived from the sequence of human erythropoietin or proteins that are homologous of human erythropoietin.

FIELD OF THE INVENTION

The present invention relates to compounds that are capable ofstimulating neuronal differentiation, neurite outgrowth and survival ofneural cells, and enhancing synaptic plasticity, learning and memory,methods of treating diseases and conditions of nervous system byadministration of compositions comprising said compounds. The compoundsand compositions of the invention include peptides and/or peptidemultimers that derived from human erythropoietin or proteins that arehomologous of human erythropoietin.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a glycoprotein hormone produced by the kidney inresponse to tissue hypoxia that stimulates red blood cell production inthe bone marrow. The gene for erythropoietin has been cloned andexpressed in Chinese hamster ovary (CHO) cells as described in U.S. Pat.No. 4,703,008. Recombinant human erythropoietin (r-HuEPO or Epoetinalfa) has an amino acid sequence identical to that of human urinaryerythropoietin, and the two are indistinguishable in chemical, physicaland immunological tests. Recombinant human erythropoietin acts byincreasing the number of cells capable of differentiating into matureerythrocytes, triggering their differentiation and augmenting hemoglobinsynthesis in developing erythroblasts (Krantz S B. Blood (1991) 77:419-434, Beckman B S, Mason-Garcia M. The Faseb Journal (1991) 5:2958-2964).

Epoetin alfa has been well tolerated in studies conducted to date.Hypertensive encephalopathy and seizures have occasionally beendescribed in dialysis patients treated with Epoetin alfa, particularlyduring the early phase of therapy when hematocrit is rising. (Eschbach JW, Egrie J C, Downing M R, Browne J K. Adamson J W. New Engl J Med(1987) 316: 73-78, Winearls C G. Oliver D O, Pippard M J, et al. Lancet(1986) 2 (8517): 1175-1177). Such reports became more rare as experienceof use of the compound developed. Occasionally, cancer patients treatedwith Epoetin alfa have experienced an increase in blood pressureassociated with a significant increase in hematocrit. The risk, however,appears substantially lower than in chronic renal failure patients.

No antibody titers against Epoetin alfa could be demonstrated andconfirmed in subjects treated with Epoetin alfa for up to 2 years,indicating the absence of immunological sensitivity to Epoetin alfa.Skin rashes and urticaria have been observed rarely and when reportedhave been mild and transient in nature, but these events suggestallergic hypersensitivity to some components of the Epoetin alfaformulation.

Epoetin an is approved for sale in many countries for the treatment ofanemia in chronic renal failure (dialysis and predialysis), anemia inzidovudine treated HIV positive patients (US), anemia in cancer patientsreceiving platinum-based chemotherapy, as a facilitator of autologousblood pre-donation, and as a peri-surgical adjuvant to reduce thelikelihood of requiring allogeneic blood transfusions in patientsundergoing orthopedic surgery.

EPO influences neuronal stern coils, likely during embryonicdevelopment, and possibly during in vitro experiments ofdifferentiation. (Juul et al Pediatr Dev Pathol (1999) 2(2) 148-158.Juul et al Pediatr Res (1998) 43(1) 40-49.) Further, neonates andinfants that suffer CNS injury via hypoxia, meningitis, andintraventricular hemorrhage, show an EPO induced neuroprotective effect(Juul et al Pod Res (1999) 46(5) 543-547.)

EPO helps prevent apoptosis of neural tissue in cases of injury thatcreate hypoxia. This may be the result of EPO produced locally byastrocytes (Morishita et al Neuroscience (1996) 76(1) 105-116).Neuroprotection has been demonstrated in gerbil hippocampal and ratcerebrocortical tissue (Sakanaka et al PNAS (1998) 95(8) 4635-4640.Sadamoto et al Biochem Biophys Res Commun (1998) 253(1) 26-32).

EPO induces biological effects of PC12 cells, including changes inCa<2+>, changes in membrane potential, and promotion of neuronalsurvival. This has been interpreted that EPO can stimulate neuralfunction and viability (Koshimura et al J. Neurochem (1999) 72(6)2565-2572. Tabria et al Int J Dev Neurosci (1995) 13(3/4) 241-252.).

A number of studies were attempted to associated diverse biologicalactivities of EPO with the particular structural areas of the protein(Grodberg et al, Eur J Biochem (1993) 218(2):597-601; Wen et al. J BiolChem (1994) 269(38):22839-22846; Elliott et al. Blood (1997)89(2):493-602; Cheetham et al. Nat Str Biol (1998) 5(10):861-866; Syedet al. Nature (1998) 395:516) Campana et al proposed that a 17 aminoacid peptide sequence of EPO can act through the EPO-R (Erythropoietinreceptor) to induce biological activity in NS20Y. SK-N-MC, and PC12cells, which includes sprouting, differentiation and neuroprotection.Curiously this peptide does not promote proliferation of hematologiccells, thus it appears inactive in cell lines well understood for theirsensitivity to EPO activity (Campana et al Int J Mol Med (1998) 1(1)235-241).

Short peptide fragments of Epo or other peptide sequences that have thefull range of biological activity of human erythropoietin or onlycertain biological activities of erythropoietin are of great interest asdrug candidates and a number of patent applications has alreadydescribed or contemplated the uses of such biologically active peptidefragments as neurotrofic or neuroprotective drugs (U.S. Pat. Nos.5,700,909, 6,703,480, 6,642,363, 5,106,954, US2003130197). The presentapplication provides new peptide fragments derived from Epo or Epofunctional homologues that may be advantageously used in therapeutictreatment of diseases or conditions of neural system.

SUMMARY OF INVENTION

The present invention identifies a new group of peptide sequences whichare potent stimulators of neurite outgrowth. Surprisingly, the sequencesare also capable to protect neuronal cells from death and promotesurvival of said cells, and moreover, are capable of stimulating cellproliferation. The inventors identified the structural motif thatpresent in all disclosed herein peptide sequences and correlated thepresence of this motif with biological activity of the sequences.

Accordingly, in the first aspect the invention relates to a compoundcomprising at least one isolated peptide sequence of 6 to 25 amino acidresidues comprising the amino acid sequence motif of the formula

(SEQ ID NO: 43) x¹-x²-x³-x⁴-x⁵-x⁶,

-   -   wherein    -   x¹ is a charged amino acid residue,    -   x⁶ is a hydrophobic amino acid residue or A, and    -   x², x³, x⁴ and x⁵ is any amino acid residue

The invention discloses a group of particular sequences comprising theabove motif, wherein said sequences are either short peptide fragmentsof human erythropoietin or short fragments of proteins having astructural homology to human erythropoietin. The disclosed sequencespossess neuritogenic activity, neuronal cell survival promotingactivity, synaptic plasticity stimulating activity, and/or learning andmemory stimulating activity and cell proliferation stimulating activity.

In another aspect the invention relates to use of a peptide sequencecomprising the structural motif of above and/or a compound comprisingsuch sequence for

-   -   the stimulating neurite outgrowth and/or promoting survival of        neural cells,    -   the manufacture of a medicament for treatment of conditions of        the central and peripheral nervous system,    -   the manufacture of a medicament for the stimulation of the        ability to learn and/or the short and/or long-term memory    -   the production of an antibody capable of recognizing an epitope        comprising the sequence;    -   the treatment an individual in need.

The invention also related to a pharmaceutical composition comprising acompound and/or peptide sequence of the invention.

In further aspect the invention relates to an antibody capable ofrecognizing the epitope comprising a sequence comprising the motif ofthe invention.

FIGURE LEGENDS

FIG. 1 Effect of recombinant human erythropoietin (Epo) on neuriteoutgrowth from CGN

FIG. 2 Effect of Epo1 (Epo-peptide 1) (SEQ ID NO: 1) on neuriteoutgrowth from CGN

FIG. 3 Effect of Epo2 (Epo-peptide 2) (SEQ ID NO: 2) on neuriteoutgrowth from CGN in vitro.

FIG. 4 Effect of Epo3 (Epo-peptide 3) (SEQ ID NO: 3) on neuriteoutgrowth from CGN in vitro.

FIG. 5 Effect of Epo4 (Epo-peptide 4) (SEQ ID NO; 4) on neuriteoutgrowth from CGN in vitro.

FIG. 6 Effect of recombinant human erythropoietin (Epo) on survival ofCGN in vitro.

FIG. 7 Effect of Epo1 (Epo-peptide 1) (SEQ ID NO: 1) on survival of CGNin vitro.

FIG. 8 Effect of Epo2 (Epo-peptide 2) (SEQ ID NO; 2) on survival of CGNin vitro.

FIG. 9 Effect of Epo3 (Epo-peptide 3) (SEQ ID NO: 3) on survival of CGNin vitro.

FIG. 10 Effect of Epo4 (Epo-peptide 4) (SEQ ID NO: 4) on survival of CGNin vitro.

FIG. 11 Effect of Epo1 (Ep1) (SEQ ID NO: 1) on proliferation of humanerythroleukemia cells TF-1 in vitro. Statistical significance wascalculated in comparison with control.

FIG. 12 Effect of Epo2 (Ep2) (SEQ ID NO: 2) on proliferation of humanerythroleukemia cells TF-1 in vitro. Statistical significance wascalculated in comparison with control.

FIG. 13 Effect of Epo3 (Ep3) (SEQ ID NO:3) on proliferation of humanerythroleukemia cells TF-1 in vitro. Statistical significance wascalculated in comparison with control.

FIG. 14 Effect of Epo4 (Ep4) (SEQ ID NO: 4) on proliferation of humanerythroleukemia cells TF-1. Statistical significance was calculated incomparison with control.

FIG. 15 Effect of recombinant human erythropoietin (EPO) and EPO mimeticpeptides Epo2, Epo3 and Epo 4 (EP2, EP3 and EP4) (SEQ ID NO: 2, 3 and 4)on the hematocrit in rat brain after the brain lesion. The hematocrit(%) is significantly increased after treatment of the rats with EPO(63.7+−1.9 EPO vs 48.7+−3.35 control, p<0.02). The peptide mimetics haveno statistically significant effect on the hematocrit (Ep2, Ep3, Ep4 vscontrol p>0.02). Control—treatment with water.

FIG. 16 Binding of recombinant erythropoietin receptor (EPOR) to theimmobilized Epo3 peptide (SEQ ID NO:3). The binding was studied by SPRanalysis. Three independent experiments were performed. The results showthat EPOR binds to Epo3 with relatively middle association rate(k_(a)=2.1*10⁴+−1.4*10⁴) and slow dissociation rate(k_(d)=4.33*10⁻⁵+−1.62*10⁻⁵). The EPOR binds with Epo3 with overallbinding affinity 4.48 nM.

-   -   A—binding of soluble EPOR to Epo3 ionic bound to the chip    -   B—binding of soluble EPOR to Epo3 covalently bound to the chip.

DETAILED DESCRIPTION OF THE INVENTION

Molecules with the potential to promote neurite outgrowth as well asstimulate survival, regeneration of neuronal cells, such as certainendogenous trophic factors, are prime targets in the search forcompounds that facilitate for example neuronal regeneration and otherforms of neuronal plasticity. Short peptide sequences of 6-25 amino acidresidues have proved to be good candidate compounds useful as inresearch as in medical applications.

Peptide Sequences

Thus, in one aspect the invention relates to a compound, whichcomprises, essentially comprises or consists of at least one isolatedpeptide sequence comprising the amino acid sequence motif of the formula

(SEQ ID NO: 43) x¹-x²-x³-x⁴-x⁵-x⁶,

-   -   wherein    -   x¹ is a charged amino acid residue,    -   x⁶ is a hydrophobic amino acid residue or A,    -   and    -   x², x³, x⁴ and x⁵ is any amino acid residue

Beyond the presence of two most important amino acid residues in thestructural motif of the invention, which are a charged residue inposition 1 (x¹) of the motif, preferably the negatively charged residue,and a hydrophobic amino acid residues in position 6 (x⁶), preferably L,V or Y, the invention further favours the sequences wherein the motiffurther comprises i) the S residue in position 2 (x²) and/or ii) ahydrophobic residue in position 2 (x²) and/or hydrophobic residue inposition 3 (x³) of the motif. Examples of such preferred motifs may bethe sequences (i) R—S-x³-x⁴-x⁵-L (SEQ ID NO:44), and (ii) R-V-x³-x⁴-x⁵-A(SEQ ID NO:45), R-V-L-x⁴-x⁵-Y (SEQ ID NO:46), K-A-V-x⁴-x⁵-L (SEQ IDNO:47), R-x²-L-x⁴-x⁵-L (SEQ ID NO:48), or R-S-L-x⁴-x⁵-L (SEQ ID NO:49).Yet, the residue S or T is in some cases preferred in position 4 (x⁴)independently of the presence of a hydrophobic residue in position x²and/or x³.

In the present application the standard one-letter code for amino acidresidues as well as the standard three-letter code are applied.Abbreviations for amino acids are in accordance with the recommendationsin the IUPAC-IUB joint Commission on Biochemical Nomenclature Eur. J.Biochem, 1984, vol. 184, pp 9-37. Throughout the description and claimseither the three letter code or the one letter code for natural aminoacids are used. Where the L or D form has not been specified it is to beunderstood that the amino acid in question has the natural L form, cf.Pure & Appl. Chem. Vol. (56(5) pp 595-624 (1984) or the D form, so thatthe peptides formed may be constituted of amino acids of L form, D form,or a sequence of mixed L forms and forms.

Where nothing is specified it is to be understood that the C-terminalamino acid of a peptide of the invention exists as the free carboxylicacid, this may also be specified as “—OH”. However, the C-terminal aminoacid of a compound of the invention may be the amidated derivative,which is indicated as “—NH₂”. Where nothing else is stated theN-terminal amino acid of a polypeptide comprise a free amino-group, thismay also be specified as “H-”.

Where nothing else is specified amino acid can be selected from anyamino acid, whether naturally occurring or not, such as alfa aminoacids, beta amino acids, and/or gamma amino acids. Accordingly, thecroup comprises but are not limited to: Ala, Val, Leu, Ile, Pro, Phe,Trp, Met, Gly, Ser, Thr, Cys, Tyr, Asn, Gln, Asp, Glu, Lys, Arg, HisAib, Nal, Sar, Orn, Lysine analogues, DAP, DAPA and 4Hyp.

Also, according to the invention modifications of the compounds/peptidesmay be performed, such as for example glycosylation and/or acetylationof the amino acids.

Basic amino acid residues are according to invention represented by theresidues of amino acids Arg, Lys, and His, acidic amino acid residues—bythe residues of amino acids Glu and Asp. Basic and amino acid residuesconstitute a group of charged amino acid residues. The group ofhydrophobic amino acid residues is represented by the residues of aminoacids Leu, Ile, Val, Phe, Trp, Tyr, and Met.

In one embodiment variants may be understood as exhibiting amino acidsequences gradually differing from the preferred predetermined sequence,as the number and scope of insertions, deletions and substitutionsincluding conservative substitutions increase. This difference ismeasured as a reduction in homology between the predetermined sequenceand the variant.

The invention relates to naturally occurring, synthetically/recombinantprepared peptide sequence/fragments, and/or peptide sequence/fragmentsprepared by means of enzymatic/chemical cleavage of a biggerpolypeptide, wherein said peptide sequence/fragments are integral partsof the polypeptide chain, said sequences being the existingseparated/isolated individual chemical units/compounds.

The present invention identifies herein a group of short individualpeptide fragments which comprise the amino acid sequence motif of aboveand correlates the presence of the motif in the sequence with thecapability of said sequence to i) induce neurite outgrowth, and/or ii)stimulate survival of neural cells, and/or stimulate synapticplasticity, and/or iv) stimulate learning, and/or v) stimulate memoryand vi) stimulate cell proliferation. This group of peptide fragmentsconsists of the following sequences:

(SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2) NENITVPDTKVNFYAWKR(SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO: 4) RVYSNFLRGKLKLYTGEA(SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 6) PSTQPWEHVNAIQEARR(SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 8) SERIDKQIRYILDGIS(SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 10) SSCLMDRHDFGFPQEEFDGNQ(SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN (SEQ ID NO: 12)CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 13) PTPVLLPAVDFSLGEWKTQM(SEQ ID NO: 14) NETVEVISE (SEQ ID NO: 15) NKNINLDSADGMPVASTD(SEQ ID NO: 16) AENNLNLPKMAEKD (SEQ ID NO: 17) ENNLRRPNLEAFNRAVKS(SEQ ID NO: 18) QQIFNLFTTKDSSAAWDE (SEQ ID NO: 19) DRMNFDIPEEIKQLQQFQK(SEQ ID NO: 20) ADNGTLFLGILKNWKEESDR (SEQ ID NO: 21)TAHKDPNAIFLSFQHLLRGKVRFL (SEQ ID NO: 22) QTRLELYKQGLRGSLTKLKGPLTM(SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK (SEQ ID NO: 24) EEQARAVQMSTKVLIQ(SEQ ID NO: 25) HIKDGDWNEFRRKLTFYLKT (SEQ ID NO: 26)LMNADSILAVKKYFRRITLY (SEQ ID NO: 27) KLEKEDFTRGKLMSSLHLKR(SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD (SEQ ID NO: 29) PNRTSGLLETNFTAS(SEQ ID NO: 30) KDFLLVIPFDCWEPVQE (SEQ ID NO: 31) ELSQWTVRSIHDLRFISS(SEQ ID NO: 32) RSFKEFLQSSLR (SEQ ID NO: 33) FINRLTGYLRN (SEQ ID NO: 34)ELSPAAKTGKR (SEQ ID NO: 35) SLIIGFAAGALYWKKRQPSL (SEQ ID NO: 36)DELINIIDGVLRDDDKNND (SEQ ID NO: 37) RNRVTNNVKDVTKLV (SEQ ID NO: 38)DKLVNIVDDLVECVKE (SERQ ID NO: 39) GLDKNTVHDQEHIMEHLEGV (SEQ ID NO: 40)SETSDCVVSSTLSPEKDSRV (SEQ ID NO: 41) QLHYFKMHDYDGNNLL.

The identified above peptides according to the application may used fordifferent applications, for example for production of differentantibodies or production of different medicaments. Therefore indifferent embodiments the peptide sequence may be preferably selected.

Thus, in one preferred embodiment, the peptide fragment may bepreferably selected from the group consisting of the peptide sequences

(SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2) NENITVPDTKVNFYAWKR(SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO: 4) RVYSNFLRGKLKLYTGEA.

The above group of sequences is designated herein as Group 1. Thesequences of Group 1 are derived from human erythropoietin (SwissprotAss. No. P01588).

In another preferred embodiment, the peptide fragment may be selectedfrom the group consisting of the peptide sequences

(SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 13) PTPVLLPAVDFSLGEWKTQM(SEQ ID NO: 21) TAHKDPNAIFLSFQHLLRGKVRFL (SEQ ID NO: 29)PNRTSGLLETNFTAS.

The above group of sequences is designated herein as Group 2. Thesequences of Group 2 are derived from human thrombopoietin (SwissprotAss. No. P40225).

In still another preferred embodiment, the peptide fragment may beselected from the group consisting of peptide sequences

(SEQ ID NO: 6) PSTQPWEHVNAIQEARR (SEQ ID NO: 14) NETVEVISE(SEQ ID NO: 22) QTRLELYKQGLRGSLTKLKGPLTM (SEQ ID NO: 30)KDFLLVIPFDCWEPVQE.

The above group of sequences is designated herein as Group 3. Thesequences of Group 3 are derived from human granulocyte-macrophagecolony-stimulating factor (GM-CSF, Swissprot Ass. No. P04141).

In yet another preferred embodiment, the peptide fragment may beselected from for the group consisting of the peptide sequences

(SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 15) NKNINLDSADGMPVASTD(SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK (SEQ ID NO: 31)ELSQWTVRSIHDLRFISS.

The above group of sequences is designated herein as Group 4. Thesequences of Group 4 are derived from human ciliary neurotrophic factor(CNFT, Swissprot Ass. No. P26441).

In still yet another preferred embodiment, the peptide fragment may beselected from the group consisting of the peptide sequences

(SEQ ID NO: 8) SERIDKQIRYILDGIS (SEQ ID NO: 16) AENNLNLPKMAEKD(SEQ ID NO: 24) EEQARAVQMSTKVLIQ (SEQ ID NO: 32) RSFKEFLQSSLR.

The above group of sequences is designated herein as Group 5. Thesequences of Group 5 are derived from human interleukin-6 Swissprot Ass.No. P05231).

In other preferred embodiments the sequence may be selected either fromthe sequences of the group of

-   i) peptide sequences derived from human interleukin-3 (IL-3,    Swissprot Ass. No. P08700) (Group 6):

(SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 17) ENNLRRPNLEAFNRAVKS or(SEQ ID NO: 25) HIKDGDWNEFRRKLTFYLKT; or

-   ii) peptide sequences derived from human interferon alpha-1    (Swissprot Ass. No. P05231) (Group 7):

(SEQ ID NO: 10) SSCLMDRHDFGFPQEEFDGNQ (SEQ ID NO: 18) QQIFNLFTTKDSSAAWDE(SEQ ID NO: 26) LMNADSILAVKKYFRRITLY; or

-   iii) peptide sequences derived from human interferon beta (Swissprot    Ass. No P01574) (Group 8):

(SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN (SEQ ID NO: 19)DRMNFDIPEEIKQLQQFQK (SEQ ID NO: 27) KLEKEDFTRGKLMSSLHLKR (SEQ ID NO: 33)FINRLTGYLRN; or

-   iv) peptide sequences derived from human interferon gamma (Swissprot    Ass. No. P05231) (Group 9):

(SEQ ID NO: 12) CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 20)ADNGTLFLGILKNWKEESDR (SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD(SEQ ID NO: 34) ELSPAAKTGKR; or

-   v) peptide sequences derived from human cell stem factor (Swissprot    Ass. No. P21583) (Group 10):

(SEQ ID NO: 37) RNRVTNNVKDVTKLV (SEQ ID NO: 38) DKLVNIVDDLVECVKE(SEQ ID NO: 40) SETSDCVVSSTLSPEKDSRV (SEQ ID NO: 35)SLIIGFAAGALYWKKRQPSL; or

-   vi) peptide sequences derived from human multiple coagulation factor    deficiency protein 2 (neural stem cell derived neuronal survival    protein/MCD2 (Swissprot Ass. No. P21583Q8NI22) (Group 11):

(SEQ ID NO: 39) GLDKNTVHDQEHIMEHLEGV (SEQ ID NO: 41) QLHYFKMHDYDGNNLL(SEQ ID NO: 36) DELINIIDGVLRDDDKNND.

However, any amino acid sequence other then the above mentionedsequences, which contains the motif of the invention and is capable ofat least one biological activity selected from stimulating neuronaldifferentiation, stimulating neurite outgrowth, stimulating survival ofneural cells, enhancing synaptic plasticity, stimulating learning andmemory is in the scope of protection of the invention.

Compounds, which comprise or consist of variants of the above sequences,are also in the scope of the invention.

“Variant of a peptide sequence” means that the peptides may be modified,for 5, example by substitution of one or more of the amino acidresidues. Both L-amino acids and D-amino acids may be used. Othermodification may comprise derivatives such as esters, sugars, etc.Examples are methyl and acetyl esters.

Variants of the peptide fragments according to the invention maycomprise, within the same variant, or fragments thereof or amongdifferent variants, or fragments thereof, at least one substitution,such as a plurality of substitutions introduced independently of oneanother. Variants of the complex, or fragments thereof may thus compriseconservative substitutions independently of one another, wherein atleast one glycine (Gly) of said variant, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Ala, Val, Leu, and Ile, and independently thereof,variants, or fragments thereof, wherein at least one alanine (Ala) ofsaid variants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Gly, Leu, and Ile,and independently thereof, variants, or fragments thereof, wherein atleast one valine (Val) of said variant, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Gly. Leu, and Ile, and independently thereof, variants, orfragments thereof, wherein at least one leucine (Leu) of said variant,or fragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Gly, Ala, Val, and Ile, andindependently thereof, variants, or fragments thereof, wherein at eastone isoleucine (Ile) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Gly, Ala, Val and Leu, and independently thereof,variants, or fragments thereof wherein at least one aspartic acids (Asp)of said variant, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Glu, Asn, and Gln,and independently thereof, variants, or fragments thereof, wherein atleast one aspargine (Asn) of said variants, or fragments thereof issubstituted with an amino acid selected from the group of amino acidsconsisting of Asp, Glu, and Gln, and independently thereof, variants, orfragments thereof, wherein at least one glutamine (Gln) of saidvariants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Asp, Glu, and Asn,and wherein at least one phenylalanine (Phe) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Tyr, Trp, His, Pro, and preferablyselected from the group of amino acids consisting of Tyr and Trp, andindependently thereof, variants, or fragments thereof, wherein at leastone tyrosine (Tyr) of said variants, or fragments thereof is substitutedwith an amino acid selected from the group of amino acids consisting ofPhe, Trp, His, Pro, preferably an amino acid selected from the group ofamino acids consisting of Phe and Trp, and independently thereof,variants, or fragments thereof, wherein at least one arginine (Arg) ofsaid fragment is substituted with an amino acid selected from the groupof amino acids consisting of Lys and His, and independently thereof,variants, or fragments thereof, wherein at least one lysine (Lys) ofsaid variants, or fragments thereof is substituted with an amino acidselected from the group of amino acids consisting of Arg and His, andindependently thereof, variants, or fragments thereof, and independentlythereof, variants, or fragments thereof, and wherein at least oneproline (Pro) of said variants, or fragments thereof is substituted withan amino acid selected from the group of amino acids consisting of Phe,Tyr, Trp, and His, and independently thereof, variants, or fragmentsthereof, wherein at least one cysteine (Cys) of said variants, orfragments thereof is substituted with an amino acid selected from thegroup of amino acids consisting of Asp, Glu, Lys, Arg, His, Asn, Ser,Thr, and Tyr.

It thus follows from the above that the same functional equivalent of apeptide fragment, or fragment of said functional equivalent may comprisemore than one conservative amino acid substitution from more than onegroup of conservative amino acids as defined herein above. The term“conservative amino acid substitution” is used synonymously herein withthe term “homologous amino acid substitution”.

The groups of conservative amino acids are as the following:

-   P, A, G (neutral, weakly hydrophobic),-   S, T (neutral, hydrophilic)-   Q, N (hydrophilic, acid amine)-   E, D (hydrophilic, acidic)-   H, K, R (hydrophilic, basic)-   A, L, I, V, M, F, Y, W (hydrophobic, aromatic)-   C (cross-link forming)

Conservative substitutions may be introduced in any position of apreferred predetermined peptide of the invention or fragment thereof. Itmay however also be desirable to introduce non-conservativesubstitutions, particularly, but not limited to, a non-conservativesubstitution in any one or more positions.

A non-conservative substitution leading to the formation of afunctionally equivalent fragment of the peptide of the invention wouldfor example differ substantially in polarity, for example a residue witha non-polar side chain (Ala, Leu, Pro, Trp, Val, Ile, Leu, Phe or Met)substituted for a residue with a polar side chain such as Gly, Ser, Thr,Cys, Tyr, Asn, or Gln or a charged amino acid such as Asp, Glu, Arg, orLys, or substituting a charged or a polar residue for a non-polar one;and/or ii) differ substantially in its effect on peptide backboneorientation such as substitution of or for Pro or Gly by anotherresidue; and/or iii) differ substantially in electric charge, forexample substitution of a negatively charged residue such as Glu or Aspfor a positively charged residue such as Lys, His or Arg (and viceversa); and/or iv) differ substantially in steric bulk, for examplesubstitution of a bulky residue such as His, Trp, Phe or Tyr for onehaving a minor side chain, e.g. Ala, Gly or Ser (and vice versa).

Substitution of amino acids may in one embodiment be made based upontheir hydrophobicity and hydrophilicity values and the relativesimilarity of the amino acid side-chain substituents, including charge,size, and the like. Exemplary amino acid substitutions which takevarious of the foregoing characteristics into consideration are wellknown to those of skill in the art and include: arginine and lysine;glutamate and aspartate; serine and threonine; glutamine and asparagine;and valine, leucine and isoleucine.

The addition or deletion of an amino acid may be an addition or deletionof from 2 to to 6 amino acids, such as from 2 to 4 amino acids. However,additions of more than 6 amino acids, such as additions from 2 to 10amino acids, are also comprised within the present invention, in themultimeric forms additions/deletions may be made individually in eachmonomer of the multimer.

As mentioned above, a compound of the invention may comprise,essentially comprise or consist of at least one peptide sequencecontaining the motif of the invention. Accordingly, a peptide sequenceof the compound may have different length.

Thus, an isolated individual peptide sequence of the compound mayconsist of 6 or more amino acid residues. The essential amino acid motifof the invention consists of 6 amino acid residues therefore the minimallength of a peptide sequence is 6 amino acid residues. The upper limitfor the number of amino acid residues in an isolated contiguous peptidesequence of the compound may vary from 7 to 50 and some cases may extendbeyond 50 amino acid residues to 100 amino acid residues. However,sequences of 25-35 amino acid residues in length or less, such as 20-25,15-20, or 10-15 amino acid residues, are within preferred embodiments ofthe invention. Peptide sequences comprising the motif of the inventionand having the length from 7 to 15 amino acid residues are referredherein as the sequences essentially comprising the motif of theinvention.

Thus, the invention preferably features compounds consisting of orcomprising at least one contiguous peptide sequence of 6-25 amino acidresidues. Thus in one embodiment, a peptide sequence may be from 10 to25, such as for example from 14 to 25, in another embodiment a sequencemay have the length from 14 to 20, for example from 14 to 18 amino acidresidues. In some embodiments, the peptide sequence of the invention maycomprise more then 25 amino acid residues, such as from 26 to 50 aminoacid residues, for example 28-30, 31-35, 36-40, 41-45 or 46-49 aminoacid residues, and in some cases the sequence may extends beyond 50amino acid residues, for example such as a sequence having the length of51-55, 56-60, 61-65, 66-71 or 72-75 amino acid residues, or 75-85 aminoacid residues.

A compound of the invention may comprise more then one of the abovepeptide sequences. Thus, the sequences of above may be formulated asmonomers, which mean that they may be represented by a single copy of anindividual peptide sequence. A compound may also comprise more then onecopy of the same sequence. Thus, the invention also relates to polymersof individual peptide sequences of the above. A polymer of a peptidesequence may be formulated as a contiguous peptide chain wherein anindividual peptide sequence is repeated/copied two or more times, or itmay a molecule, wherein the copies of an individual peptide sequence areconnected to each other then the peptide bond, for example via any kindof linker grouping. An example of such polymers may be dendromericpolymers wherein the individual copies of a peptide sequence areattached to a core molecule, such as for example a lysine residue.Another example of a polymer may be the LPA (Ligand Presenting Assembly)type polymer. Polymerisation such as repetitive sequences or attachmentto various carriers are well-known in the art. e.g. lysine backbones,such as lysine dendrimers carrying 4 peptides, 8 peptides, 16 peptides,or 32 peptides. Other carriers may be protein moieties, such as bovineserum albumin (BSA), or lipophilic dendrimers, or micelle-like carriersformed by lipophilic derivatives, or starburst (star-like) carbon chainpolymer conjugates, or ligand presenting assembly (LPA) based onderivatives of diethylaminomethane.

A compound of the invention may comprise or consists of two or moreindividual peptide fragments having different amino acid sequencespresented as isolated peptide sequence.

Proteins and Peptide Fragments Thereof

According to the invention an isolated peptide sequence which comprises,essentially comprises or consists of the motif described above is oneembodiment derived from human erythropoietin (EPO). The inventionrelates to human erythropoietin having the sequence identified in theSwissprot database under ID No. P01588. In another embodiment thepeptide sequence is derived from a variant or a homologue of humanerythropoietin.

Thus, the invention relates to proteins, sequences of which havehomology to the sequence of erythropoietin (Swissprot Ass. No. P01588).The invention defines a protein as being a homologue of humanerythropoietin when the amino acid of said protein shares at least 35%homology with the sequence of erythropoietin (Swissprot Ass. No.P01588). The homology of one amino acid sequence with another amino acidsequence is defined as a percentage of identical amino acids in the twocollated sequences. The homology between amino acid sequences may becalculated using well known algorithms such as BLOSUM 30, BLOSUM 40,BLOSUM 45, BLOSUM 50, BLOSUM 55, BLOSUM 60, BLOSUM 62, BLOSUM 65, BLOSUM70, BLOSUM 75, BLOSUM 80, BLOSUM 85, or BLOSUM 90.

Preferably the invention relates to human erythropoietin structuralhomologues selected from

thrombopoietin (Swissprot Ass. No. 82% homology to erythropoitin,P40225), granulocyte-macrophage colony- 63% homology to erythropoitin,stimulating factor (GM-CSF, Swissprot Ass. No. P04141) ciliaryneurotrophic factor (CNFT, 41% of homology to erythropoitin, SwissprotAss. No. P26441) interleukin-6 (IL-6, Swissprot Ass. 45% homology toerythropoitin, No. P05231) interleukin-3 (IL-3, Swissprot Ass. 36%homology to erythropoitin, No. P08700) interferon alpha-1 (SwissprotAss. 57% homology to erythropoitin, No. P05231) interferon beta(Swissprot Ass. 59% homology to erythropoitin, No. P01574) interferongamma (Swissprot Ass. 41% homology to erythropoitin, No. P05231) cellstem factor (Swissprot Ass. 37% homology to erythropoitin, No. P21583)multiple coagulation factor deficiency 38% homology to erythropoitin.protein 2 (MCD2, Swissprot Ass. No. Q8NI22)

According to the invention the above proteins are both structured andfunctional homologues of human erythropoietin.

An amino acid sequence, which is 35-100%, preferably 45-100%, morepreferably 50-100%, such as about 55, 60, 65, 70, 75, 80% homologous tothe sequence of erythropoietin of Swissprot Ass. No. P01588, andpossesses at least one biological activity of said erythropoietin isregarded by the application as a functional homologue of erythropoietin.Non-limited examples of functional homologues of human erythropoietinare given above. Examples of the biological activities of humanerythropoietin relevant for the present application include, but notlimited by stimulation of proliferation, differentiation and survival ofhaemopoietic cells, stimulating neuronal cell differentiation andsurvival. In a preferred embodiment the application relates tobiological activities, which are most relevant for neural coils, such asthe capability of stimulating neuronal differentiation, for examplestimulating neurite outgrowth, stimulating neuronal survival, such asinhibiting apoptosis of neural cells, and/or enhancing synapticplasticity, learning and memory. These biological functions oferythropoietin are according to the invention to be executed by anarea(s) of the protein, which essentially comprise a sequence(s)

(SEQ ID NO: 1) DSRVLERYLLEAKE (SEQ ID NO: 2) NENITVPDTKVNFYAWKR(SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO: 4) RVYSNFLRGKLKLYTGEA.

The invention also contemplates variants of human erythropoietin havingthe sequence of Swissprot Ass. No. P01588 as functional homologues ofthe protein “Variant” in the present content means i)erythropoietin-like polypeptides of non-human origin, such as otherspecies origin, ii) recombinant molecules of human erythropoietin; iii)molecules of i) and which comprise amino acid substitutions ormodifications of amino acids in relation to the sequence of Ass. No.P01588, iv) polypeptides that share 90-99% homology with the sequence ofAss. No. P01588, v) fragments of the sequence of Ass. No. P01588, whichare of 90-99% of the sequence length, or vi) natural or recombinanthuman erythropoietin molecules which have post-translationalmodifications, such as for example additional/alternative glycosylationof the polypeptide chain when compared to naturally occurringerythropoietin polypeptides.

The sequences derived from human erythropoietin identified as SEQ IDNOS: 1-4 all

-   1) comprise the motif of the invention

(SEQ ID NO: 43) x¹-x²-x³-x⁴-x⁵-x⁶,

-   -   wherein    -   x¹ is a charged amino acid residue,    -   x⁶ is a hydrophobic amino acid residue or A,    -   x², x³, x⁴ and x⁵ is any amino acid residue, and

-   2) possess neuritogenic activity,

-   3) possess survival promoting activity,

-   4) possess synaptic plasticity enhancing activity, and/or

-   5) possess learning and memory enhancing activity, and

-   6) possess cell proliferation stimulating activity

The invention considers the presence of the motif of the invention in apeptide sequence to be essential structural element to enable thepeptide sequence to induce neuronal differentiation, such as stimulateneurite growth, promote survival of neuronal cells and/or enhancesynaptic plasticity, learning capability and improve memory.Accordingly, any peptide sequence of at least 6 amino acids comprisingthe above motif, having about 40-99% structural homology to SEQ ID NO:1, 2, 3 or 4 and having at least one activity of the SEQ ID NO: 1, 2, 3or 4 is considered to be a functional homologue of SEQ ID NOS: 1, 2, 3and 4, Homology between two peptide sequences is defined as describedabove.

As described above, the invention preferably relates to short contiguouspeptide sequences of 6 to 25 amino acids long comprising the motif ofabove. The invention further prefers those of the latter peptidesequences that have at least 40% homology to any of identified abovefragments of EPO (SEQ ID NO; 1, 2, 3 or 4). More preferred the sequenceshaving about 50-55, 55-60 or 60-65% homology. Yet, more preferred thesequences which are more then 65% homologous to any of the EPO peptidefragments identified herein as SEQ ID NOs: 1-4 Homology betweensequences is identified as described above. However, in some embodimentsa sequence which has homology to a particular sequence selected from SEQID NOS: 1, 2, 3 or 4, may be preferred. The invention discloses hereinthat the sequences of SEQ ID NOS: 1-4 vary in their activity in relationto stimulating of neurite outgrowth, neural cell survival and cellproliferation, therefore, depending on the purpose it may be favourableto select a particular sequence to achieve the maximal effect of thesequence on neurite outgrowth, cell survival or cell proliferation.

Thus, in one embodiment of the invention, such preferred sequence may bethe sequence of SEQ ID NO: 1 or a sequence homologous to SEQ ID NO: 1,which may be selected form the group consisting of the sequences

(SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 6) PSTQPWEHVNAIQEARR(SEQ ID NO: 7) CSRSIWLARKIRSD (SEQ ID NO: 8) SERIDKQIRYILDGIS(SEQ ID NO: 9) SCNMIDEIITHLKQ (SEQ ID NO: 10) SSCLMDRHDFGFPQEEFDGNQ(SEQ ID NO: 11) MSYNLLGFLQRSSNFQCQKLLWQLN (SEQ ID NO: 12)CYCQDPYVKEAENLKKYFNA (SEQ ID NO: 37) RNRVTNNVKDVTKLV (SEQ ID NO: 39)GLDKNTVHDQEHIMEHLEGV

The sequences homologous to SEQ ID NO: 2 may preferably be selected formthe group consisting of the sequences:

(SEQ ID NO: 3) QLHVDKAVSGLRSLTTLLRA (SEQ ID NO: 4) RVYSNFLRGKLKLYTGEA.(SEQ ID NO: 5) DLRVLSKLLRDSHV (SEQ ID NO: 13) PTPVLLPAVDFSLGEWKTQM(SEQ ID NO: 14) NETVEVISE (SEQ ID NO: 15) NKNINLDSADGMPVASTD(SEQ ID NO: 16) AENNLNLPKMAEKD (SEQ ID NO: 17) ENNLRRPNLEAFNRAVKS(SEQ ID NO: 18) QQIFNLFTTKDSSAAWDE (SEQ ID NO: 19) DRMNFDIPEEIKQLQQFQK(SEQ ID NO: 20) ADNGTLFLGILKNWKEESDR.

The sequences homologous to SEQ ID NO: 3 may preferably be selected formthe group consisting of the sequences:

(SEQ ID NO: 21) TAHKDPNAIFLSFQHLLRGKVRFL (SEQ ID NO: 22)QTRLELYKQGLRGSLTKLKGPLTM (SEQ ID NO: 23) LLQVAAFAYQIEELMILLEYK(SEQ ID NO: 24) EEQARAVQMSTKVLIQ (SEQ ID NO: 25) HIKDGDWNEFRRKLTFYLKT(SEQ ID NO: 26) LMNADSILAVKKYFRRITLY (SEQ ID NO: 27)KLEKEDFTRGKLMSSLHLKR (SEQ ID NO: 28) NSNKKKRDDFEKLTNYSVTD(SEQ ID NO: 40) SETSDCVVSSTLSPEKDSRV (SEQ ID NO: 41) QLHYFKMHDYDGNNLL.

A sequence homologous to SEQ ID NO: 4 may preferably be selected formthe group consisting of the sequences:

(SEQ ID NO: 29) PNRTSGLLETNFTAS (SEQ ID NO: 30) KDFLLVIPFDCWEPVQE(SEQ ID NO: 31) ELSQWTVRSIHDLRFISS (SEQ ID NO: 32) RSFKEFLQSSLR(SEQ ID NO: 33) FINRLTGYLRN (SEQ ID NO: 34) ELSPAAKTGKR (SEQ ID NO: 35)SLIIGFAAGALYWKKRQPSL (SEQ ID NO: 36) DELINIIDGVLRDDDKNND

The sequences of SEQ ID NOs: 5-41 are derived from the amino acidsequences of the following human EPO homologues

-   thrombopoietin (Swissprot Ass. No. P40225),-   granulocyte-macrophage colony-stimulating factor (GM-CSF, Swissprot    Ass. No. 04141),-   neurotrophic factor (CNFT, Swissprot Ass. No. P20441),-   interleukin-6 (IL-6, Swissprot Ass. No. P05231),-   interleukin-3 (IL-3, Swissprot Ass. No. P08700),-   interferon alpha-1 (Swissprot Ass. No. P05231),-   interferon beta (Swissprot Ass. No. P01574),-   interferon gamma (Swissprot Ass. No. P05231).-   cell stem factor (Swissprot Ass. No. P21583)-   multiple coagulation factor deficiency protein 2 (MCD2, Swissprot    Ass. No. Q8NI22)

The term “derived” in relation to an individual peptide sequence meansthat said peptide sequence is originally an integral part/fragment of anaturally occurring polypeptide/protein which has been prepared as anisolated individual chemical entity by means of biochemical or chemicalmethods, for example by enzymatic cleavage of the original polypeptideor synthetic preparation of the peptide sequence. The present patentapplication relates to peptide sequences derived from the aboveidentified proteins as isolated individual peptide fragments of soldproteins, and does not relate to the original proteins/polypeptidessequences of which include these fragments.

Individual peptide sequences/fragments of the invention possessbiological activity(s) that can mimic certain biological activity(s) ofthe original polypeptide. Thus, the individual peptide sequence/fragmentof the invention may be used as a functional equivalent/homologue of thepredetermined polypeptide selected from the above identified, or afunctional equivalent of human EPO or a biological fragment thereof.

Production of Individual Peptide Sequences

The peptide sequences of the present invention may be prepared by anyconventional synthetic methods, recombinant DNA technologies, enzymaticcleavage of full-length proteins which the peptide sequences are derivedfrom, or a combination of said methods.

Recombinant Preparation

Thus, in one embodiment the peptides of the invention are produced byuse of recombinant DNA technologies.

The DNA sequence encoding a peptide or the corresponding full-lengthprotein the peptide originates from may be prepared synthetically byestablished standard methods, e.g. the phosphoamidine method describedby Beaucage and Caruthers, 1981, Tetrahedron Lett. 22:1859-1869, or themethod described by Matthes et al., 1984, EMBO J. 3:801-805. Accordingto the phosphoamidine method, oligonucleotides are synthesised, e.g. inan automatic DNA synthesiser, purified, annealed, ligated and cloned insuitable vectors.

The DNA sequence encoding a peptide may also be prepared byfragmentation of the DNA sequences encoding the correspondingfull-length protein of peptide origin, using DNAase I according to astandard protocol (Sambrook et al., Molecular cloning: A Laboratorymanual. 2 nd ed., CSHL Press, Cold Spring Harbor, N.Y., 1989). Thepresent invention relates to full-length proteins selected from thegroups of proteins identified above. The DNA encoding the full-lengthproteins of the invention may alternatively be fragmented using specificrestriction endonucleases. The fragments of DNA are further purifiedusing standard procedures described in Sambrook et al., Molecularcloning: A Laboratory manual. 2 nd ed., CSHL Press, Cold Spring Harbor,N.Y., 1989.

The DNA sequence encoding a full-length protein may also be of genomicor cDNA origin, for instance obtained by preparing a genomic or cDNAlibrary and screening for DNA sequences coding for all or part of thefull-length protein by hybridisation using synthetic oligonucleotideprobes in accordance with standard techniques (cf. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Ed., Cold Spring Harbor,1989). The DNA sequence may also be prepared by polymerase chainreaction using specific primers, for instance as described in U.S. Pat.No. 4,683,202 or Saiki et al., 1988, Science 239:487-491.

The DNA sequence is then inserted into a recombinant expression vector,which may be any vector, which may conveniently be subjected torecombinant DNA procedures. The choice of vector will often depend onthe host cell into which it is to be introduced. Thus, the vector may bean autonomously replicating vector, i.e. a vector that exists as anextrachromosomal entity, the replication of which is independent ofchromosomal replication, e.g. a plasmid. Alternatively, the vector maybe one which, when introduced into a host cell, is integrated into thehost cell genome and replicated together with the chromosome(s) intowhich it has been integrated.

In the vector, the DNA sequence encoding a peptide or a fun-lengthprotein should be operably connected to a suitable promoter sequence.The promoter may be any DNA sequence, which shows transcriptionalactivity in the host cell of choice and may be derived from genesencoding proteins either homologous or heterologous to the host cell.Examples of suitable promoters for directing the transcription of thecoding DNA sequence in mammalian cells are the SV 40 promoter (Subramaniet al., 1981, Mol. Cell Biol. 1:854-864), the MT-1 (metallothioneingene) promoter (Palmiter et al., 1983. Science 222: 809-814) or theadenovirus 2 major late promoter. A suitable promoter for use in insectcells is the polyhedrin promoter (Vasuvedan et al., 1992, FEBS Lett.311:7-11). Suitable promoters for use in yeast host cells includepromoters from yeast glycolytic genes (Hitzeman et al., 1980, J. Biol.Chem. 255:12073-12080; Alber and Kawasaki, 1982, J. Mol. Appl. Gen. 1:419-434) or alcohol dehydrogenase genes (Young et al., 1982, in GeneticEngineering of Microorganisms for Chemicals, Hollaender et al. eds.,Plenum Press, New York), or the TPI1 (U.S. Pat. No. 4,599,311) orADH2-4c (Russell et al., 1983. Nature 304:652-654) promoters. Suitablepromoters for use in filamentous fungus host cells are, for instance,the ADH3 promoter (McKnight et al., 1985, EMBO J. 4:2093-2099) or thetpiA promoter.

The coding DNA sequence may also be operably connected to a suitableterminator, such as the human growth hormone terminator (Palmiter etal., op. cit.) or (for fungal hosts) the TPI1 (Alber and Kawasaki, op.cit.) or ADH3 (McKnight et al., op. cit.) promoters. The vector mayfurther comprise elements such as polyadenylation signals (e.g. from SV40 or the adenovirus 5 Elb region), transcriptional enhancer sequences(e.g. the SV 40 enhancer) and translational enhancer sequences (e.g. theones encoding adenovirus VA RNAs).

The recombinant expression vector may further comprise a DNA sequenceenabling the vector to replicate in the host cell in question. Anexample of such a sequence (when the host cell is a mammalian cell) isthe SV 40 origin of replication. The vector may also comprise aselectable marker, e.g. a gene the product of which complements a defectin the host cell, such as the gene coding for dihydrofolate reductase(DHFR) or one which confers resistance to a drug, e.g. neomycin,hydromycin or methotrexate.

The procedures used to ligate the DNA sequences coding the peptides orfull-length proteins, the promoter and the terminator, respectively, andto insert them into suitable vectors containing the informationnecessary for replication, are well known to persons skilled in the art(cf., for instance, Sambrook et al., op. cit.).

To obtain recombinant peptides of the invention the coding DNA sequencesmay be usefully fused with a second peptide coding sequence and aprotease cleavage site coding sequence, giving a DNA construct encodingthe fusion protein, wherein the protease cleavage site coding sequencepositioned between the HBP fragment and second peptide coding DNA,inserted into a recombinant expression vector, and expressed inrecombinant host cells. In one embodiment, said second peptide selectedfrom, but not limited by the group comprising glutathion-S-reductase,calf thymosin, bacterial thioredoxin or human ubiquitin natural orsynthetic variants, or peptides thereof. In another embodiment, apeptide sequence comprising a protease cleavage site may be the FactorXa, with the amino acid sequence IEGR, enterokinase, with the amino acidsequence DDDDK, thrombin, with the amino acid sequence LVPR/GS, orAcharombacter lyticus, with the amino acid sequence XKX, cleavage site.

The host cell into which the expression vector is introduced may be anycell which is capable of expression of the peptides or full-lengthproteins, and is preferably a eukaryotic cell, such as invertebrate(insect) cells or vertebrate cells, e.g. Xenopus laevis oocytes ormammalian cells, in particular insect and mammalian cells. Examples ofsuitable mammalian cell lines are the HEK293 (ATCC CRL-1573), COS (ATCCCRL-1650), BHK (ATCC CRL-1632, ATCC CCL-10) or CHO (ATCC CCL-61) celllines. Methods of transfecting mammalian cells and expressing DNAsequences introduced in the cells are described in e.g. Kaufman andSharp, J. Mol. Biol. 159, 1982, pp. 601-621; Southern and Berg, 1982, J.Mol. Appl. Genet. 1:327-341; Loyter et al., 1982, Proc. Natl. Acad. Sci.USA 79: 422-426; Wigler et al., 1978, Cell 14:725; Corsaro and Pearson,1981, in Somatic Cell Genetics 7, p. 603; Graham and van der Eb, 1973,Virol. 52:456; and Neumann et al., 1982, EMBO J. 1:841-845.

Alternatively, fungal cells (including yeast cells) may be used as hostcells. Examples of suitable yeast cells include cells of Saccharomycesspp. or Schizosaccharomyces spp., in particular strains of Saccharomycescerevisiae. Examples of other fungal cells are cells of filamentousfungi, e.g. Aspergillus spp. or Neurospora spp., in particular strainsof Aspergillus oryzae or Aspergillus niger. The use of Aspergillus spp.for the expression of proteins is described in, e.g., EP 238 023.

The medium used to culture the cells may be any conventional mediumsuitable for growing mammalian cells, such as a serum-containing orserum-free medium containing appropriate supplements, or a suitablemedium for growing insect, yeast or fungal cells. Suitable media areavailable from commercial suppliers or may be prepared according topublished recipes (e.g. in catalogues of the American Type CultureCollection).

The peptides or full-length proteins recombinantly produced by the cellsmay then be recovered from the culture medium by conventional proceduresincluding separating the host cells from the medium by centrifugation orfiltration, precipitating the proteinaceous components of thesupernatant or filtrate by means of a salt, e.g. ammonium sulphate,purification by a variety of chromatographic procedures, e.g. HPLC, ionexchange chromatography, affinity chromatography, or the like.

Synthetic Preparation

The methods for synthetic production of peptides are well known in theart. Detailed descriptions as well as practical advice for producingsynthetic peptides may be found in Synthetic Peptides: A User's Guide(Advances in Molecular Biology), Grant G, A. ed., Oxford UniversityPress, 2002, or in: Pharmaceutical Formulation: Development of Peptidesand Proteins, Frokjaer and Hovgaard eds., Taylor and Francis, 1999.

Peptides may for example be synthesised by using Fmoc chemistry and withAcm-protected cysteines. After purification by reversed phase HPLC,peptides may be further processed to obtain for example cyclic or C- orN-terminal modified isoforms. The methods for cyclization and terminalmodification are well-known in the art and described in detail in theabove-cited manuals.

In a preferred embodiment the peptide sequences of the invention areproduced synthetically, in particular, by the Sequence Assisted PeptideSynthesis (SAPS) method.

By SAPS peptides may be synthesised either batchwise in a polyethylenevessel equipped with a polypropylene filter for filtration or in thecontinuous-flow version of the polyamide solid-phase method (Dryland, A,and Sheppard, R. C., (1986) J. Chem. Soc. Perkin Trans. I, 125-137.) ona fully automated peptide synthesiser using 9-fluorenylmethyloxycarbonyl(Fmoc) or tert.-Butyloxycarbonyl, (Boc) as N-a-amino protecting groupand suitable common protection groups for side-chain functionality.

When synthesised, individual peptide sequences may then be formulated asmultimers using well-known in the art techniques, for examples dimers ofthe sequences may be obtained by the LPA method described in WO00/18791, dendrimeric polymers by the MAP synthesis described inPCT/US90/02039.

Antibody

It is an objective of the present invention to provide an antibody,antigen binding fragment or recombinant protein thereof capable ofrecognizing and selectively binding to an epitope on humanerythropoietin, thrombopoietin, granulocyte-macrophagecolony-stimulating factor (GM-CSF), ciliary neurotrophic factor (CNFT),interleukin-6 (IL-6), interleukin-3 (IL-3), interferon alpha-1,interferon beta, interferon gamma, cell stem factor, multiplecoagulation factor deficiency protein 2 (MCD2), said epitope comprisingat least one of the sequences selected from SEQ ID NOs:1-41, or afragment of said sequence.

By the term “epitope” is meant the specific group of atoms (on anantigen molecule) that is recognized by (that antigen's) antibodies(thereby causing an immune response). The term “epitope” is theequivalent to the term “antigenic determinant”. The epitope may comprise3 or more amino acid residues, such as for example 4, 5, 6, 7, 8 aminoacid residues, located in close proximity, such as within a contiguousamino acid sequence, or located in distant parts of the amino acidsequence of an antigen, but due to protein folding have been approachedto each other.

Antibody molecules belong to a family of plasma proteins calledimmunoglobulins, whose basic building block, the immunoglobulin fold ordomain, is used in various forms in many molecules of the immune systemand other biological recognition systems. A typical immunoglobulin hasfour polypeptide chains, containing an antigen binding region known as avariable region and a non-varying region known as the constant region.

Native antibodies and immunoglobulins are usually heterotetramericglycoproteins of about 150,000 daltons, composed of two identical light(L) chains and two identical heavy (H) chains. Each light chain islinked to a heavy chain by one covalent disulfide bond, while the numberof disulfide linkages varies between the heavy chains of differentimmunoglobulin isotypes. Each heavy and light chain also has regularlyspaced intrachain disulfide bridges. Each heavy chain has at one end avariable domain (VH) followed by a number of constant domains. Eachlight chain has a variable domain at one end (VL) and a constant domainat its other end. The constant domain of the light chain is aligned withthe first constant domain of the heavy chain, and the light chainvariable domain is aligned with the variable domain of the heavy chain.Particular amino acid residues are believed to form an interface betweenthe light and heavy chain variable domains (Novotny J, & Haber E. ProcNatl Acad Sci USA, 82(14):4592-6, 1985).

Depending on the amino acid sequences of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are at least five (5) major classes of immunoglobulins IgA, IgD,IgE, IgG and several of these may be further divided into subclasses(isotypes), e.g. IgG-1. IgG-3 and IgA-1 and IgA-2. The heavy chainsconstant domains that correspond to the different classes ofimmunoglobulins are called alpha (α), delta (δ), epsilon (ε), gamma (γ)and mu (μ), respectively. The light chains of antibodies can be assignedto one of two clearly distinct types, called kappa (κ) and lambda (λ),based on the amino sequences of their constant domain. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The term “variable” in the context of variable domain of antibodies,refers to the fact that certain portions of the variable domains differextensively in sequence among antibodies. The variable domains are forbinding and determine the specificity of each particular antibody forits particular antigen. However, the variability is not evenlydistributed through the variable domains of antibodies. It isconcentrated in three segments called complementarity determiningregions (CDRs) also known as hypervariable regions both in the lightchain and the heavy chain variable domains.

The more highly conserved portions of variable domains are called theframework (FR). The variable domains of native heavy and light chainseach comprise four FR regions, largely a adopting a β-sheetconfiguration, connected by three CDRs, which form loops connecting, andin some cases forming part of, the β-sheet structure. The CDRs in eachchain are held together in close proximity by the FR regions and, withthe CDRs from the other chain, contribute to the formation of theantigen-binding site of antibodies. The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

An antibody that is contemplated for use in the present invention thuscan be in any of a variety of forms, including a whole immunoglobulin,on antibody fragment such as Fv, Fab, and similar fragments, a singlechain antibody which includes the variable domain complementaritydetermining regions (CDR), and the like forms, all of which fall underthe broad term “antibody”, as used herein. The present inventioncontemplates the use of any specificity of an antibody, polyclonal ormonoclonal, and is not limited to antibodies that recognize andimmunoreact with a specific antigen. In preferred embodiments, in thecontext of both the therapeutic and screening methods described below,an antibody or fragment thereof is used that is immunospecific for anantigen or epitope of the invention.

The term “antibody fragment” refers to a portion of a full-lengthantibody, generally the antigen binding or variable region. Examples ofantibody fragments include Fab, Fab′, F(ab′)₂ and FY fragments. Papaindigestion of antibodies produces two identical antigen bindingfragments, called the Fab fragment, each with a single antigen bindingsite, and a residual “Fc” fragment, so-called for its ability tocrystallize readily. Pepsin treatment yields an F(ab′)₂ fragment thathas two antigen binding fragments that are capable of cross-linkingantigen, and a residual other fragment (which is termed pFc′).Additional fragments can include diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragments. As used herein, “functional fragment” withrespect to antibodies, refers to Fv, F(ab) and F(ab′)₂ fragments.

The term “antibody fragment” is used herein interchangeably with theterm “antigen binding fragment”.

Antibody fragments may be as small as about 4 amino acids, 5 aminoacids, 6 amino acids, 7 amino acids, 9 amino acids, about 12 aminoacids, about 15 amino acids, about 17 amino acids, about 18 amino acids,about 20 amino acids, about 25 amino acids, about 30 amino acids ormore. In general, an antibody fragment of the invention can have anyupper size limit so long as it is has similar or immunologicalproperties relative to antibody that binds with specificity to anepitope comprising a peptide sequence selected from any of the sequencesidentified herein as SEQ ID NOs: 1-41, or a fragment of said sequences.Thus, in context of the present invention the term “antibody fragment”is identical to term “antigen binding fragment”.

Antibody fragments retain some ability to selectively bind with itsantigen or receptor. Some types of antibody fragments are defined asfollows:

-   -   (1) Fab is the fragment that contains a monovalent        antigen-binding fragment of an antibody molecule. A Fab fragment        can be produced by digestion of whole antibody with the enzyme        papain to yield an intact light chain and a portion of one heavy        chain.    -   (2) Fab′ is the fragment of an antibody molecule can be obtained        by treating whole antibody with pepsin, followed by reduction,        to yield an intact light chain and a portion of the heavy chain.        Two Fab′ fragments are obtained per antibody molecule.

Fab′ fragments differ from Fab fragments by the addition of a fewresidues at the carboxyl terminus of the heavy chain CH1 domainincluding one or more cysteines from the antibody hinge region.

-   -   (3) (Fab′)₂ is the fragment of an antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction.    -   (4) F(ab′)₂ is a dimer of two Fab′ fragments held together by        two disulfide bonds.        Fv is the minimum antibody fragment that contains a complete        antigen recognition and binding site. This region consists of a        dimer of one heavy and one light chain variable domain in a        tight, non-covalent association (V_(H)-V_(L) dimer). It is in        this configuration that the three CDRs of each variable domain        interact to define an antigen binding site on the surface of the        V_(H)-V_(L) dimer. Collectively, the six CDRs confer antigen        binding specificity to the antibody. However, even a single        variable domain (or halt of an Fv comprising only three CDRs        specific for an antigen) has the ability to recognize and bind        antigen, although at a lower affinity than the entire binding        site.    -   (5) Single chain antibody (“SCA”), defined as a genetically        engineered molecule containing the variable region of the light        chain, the variable region of the heavy chain, linked by a        suitable polypeptide linker as a genetically fused single chain        molecule. Such single chain antibodies are also referred to as        “single-chain Fv” or “sFv” antibody fragments. Generally the Fv        polypeptide further comprises a polypeptide linker between the        VH and VL domains that enables the sFv to form the desired        structure for antigen binding. For a review of sFv see Pluckthun        in The Pharmacology of Monoclonal Antibodies 113: 269-315        Rosenburg and Moore eds. Springer-Verlag, NY, 1994.

The term “diabodies” refers to a small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (VH) connected to a light chain variable domain (VL) in the samepolypeptide chain (VH-VL). By using a linker that is too short to allowpairing between the two domains on the same chain, the domains areforced to pair with the complementary domains of another chain andcreate two antigen-binding sites. Diabodies are described more fully in,for example, EP 404,097; WO 93/11161, and Hollinger et al., Proc. Natl.Acad Sci. USA 90: 6444-6448 (1993).

The invention contemplate both polyclonal and monoclonal antibody,antigen binding fragments and recombinant proteins thereof which arecapable of binding an epitope according to the invention.

The preparation of polyclonal antibodies is well-known to those skilledin the art. See, for example, Green et al. 1992. Production ofPolyclonal Antisera, in: immunochemical Protocols (Manson, ed.), pages1-5 (Humana Press); Coligan, et al., Production of Polyclonal Antiserain Rabbits, Rats Mice and Hamsters, in: Current Protocols in Immunology,section 2.4.1, which are hereby incorporated by reference.

The preparation of monoclonal antibodies likewise is conventional. See,for example, Kohler & Milstein, Nature, 256:495-7 (1975); Coligan, etal., sections 2.5.1-2.6.7; and Harlow, et al., in: Antibodies: ALaboratory Manual, page 726, Cold Spring Harbor Pub. (1938), Monoclonalantibodies can be isolated and purified from hybridoma cultures by avariety of well-established techniques. Such isolation techniquesinclude affinity chromatography with Protein-A Sepharose, size-exclusionchromatography, and ion-exchange chromatography. See, e.g., Coligan, etal., sections 2.7.1-2.7.12 and sections 2.9.1-2.9.3; Barnes, et al.,Purification of Immunoglobulin G (IgG). In: Methods in MolecularBiology, 1992, 10:79-104, Humana Press, NY.

Methods of in vitro and in vivo manipulation of monoclonal antibodiesare well known to those skilled in the art. For example, the monoclonalantibodies to be used in accordance with the present invention may bemade by the hybridoma method first described by Kohler and Milstein,1975, Nature 256, 495-7, or may be made by recombinant methods, e.g., asdescribed in U.S. Pat. No. 4,816,567. The monoclonal antibodies for usewith the present invention may also be isolated from phage antibodylibraries using the techniques described in Clackson et al., 1991.Nature 352: 624-628, as well as In Marks et al., 1991, J Mol Biol 222;58′1-597. Another method involves humanizing a monoclonal antibody byrecombinant means to generate antibodies containing human specific andrecognizable sequences. See, for review. Holmes, et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore. In contrast toconventional polyclonal antibody preparations that typically includedifferent antibodies directed against different determinants (epitopes),each monoclonal antibody is directed against a single determinant on theantigen. In additional to their specificity, the monoclonal antibodiesare advantageous in that they are synthesized by the hybridoma culture,uncontaminated by other immunoglobulins. The modifier “monoclonal”indicates the character of the antibody indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method.

The monoclonal antibodies herein specifically include “chimeric”antibodies (immunoglobulins) in which a portion of the heavy and/orlight chain is identical with or homologous to corresponding sequencesin antibodies derived from a particular species or belonging to aparticular antibody class or subclass, while the remainder of thechain(s) is identical with or homologous to corresponding sequences inantibodies derived from another species or belonging to another antibodyclass or subclass, as well as fragments of such antibodies, so long asthey exhibit the desired biological activity (U.S. Pat. No. 4,816,567);Morrison et al., 1984, Proc Natl Acad Sci 81: 6851-6855.

Methods of making antibody fragments are also known in the art (see forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, NY, 1988, incorporated herein by reference). Antibodyfragments of the present invention can be prepared by proteolytichydrolysis of the antibody or by expression in E. coli of DNA encodingthe fragment. Antibody fragments can be obtained by pepsin or papaindigestion of whole antibodies conventional methods. For example,antibody fragments can be produced by enzymatic cleavage of antibodieswith pepsin to provide a 5S fragment denoted F(ab′)₂. This fragment canbe further cleaved using a thiol reducing agent, and optionally ablocking group for the sulfhydryl groups resulting from cleavage ofdisulfide linkages, to produce 3.5S Fab′ monovalent fragments.Alternatively, an enzymatic cleavage using pepsin produces twomonovalent Fab′ fragments and an Fc fragment directly. These methods aredescribed, for example, in U.S. Pat. Nos. 4,036,945 and 4,331,647, andreferences contained therein. These patents are hereby incorporated intheir entireties by reference.

Other methods of cleaving antibodies, such as separation of heavy chainsto form monovalent light-heavy chain fragments, further cleavage offragments, or other enzymatic, chemical, or genetic techniques may alsobe used, so long as the fragments bind to the antigen that is recognizedby the intact antibody. For example, Fv fragments comprise anassociation of V_(H) and V_(L) chains. This association may benoncovalent or the variable chains can be linked by an intermoleculardisulfide bond or cross-linked by chemicals such as glutaraldehyde.Preferably, the Fv fragments comprise V_(H) and V_(L) chains connectedby a peptide linker. These single-chain antigen binding proteins (sFv)are prepared by constructing a structural gene comprising DNA sequencesencoding the V_(H) and V_(L) domains connected by an oligonucleotide.The structural gene is inserted into an expression vector, which issubsequently introduced into a host cell such as E. coli. Therecombinant host cells synthesize a single polypeptide chain with alinker peptide bridging the two V domains. Methods for producing sFvsare described, for example, by Whitlow, et al., 1991, In: Methods: ACompanion to Methods in Enzymology, 2:97; Bird et al., 1988. Science242; 423-426; U.S. Pat. No. 4,946,778; and Pack, et al., 1993,BioTechnology 11; 1271-77.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR), CDR peptides (“minimalrecognition units”) are often involved in antigen recognition andbinding. CDR peptides can be obtained by cloning or constructing genesencoding the CDR of an antibody of interest. Such genes are prepared,for example, by using the polymerase chain reaction to synthesize thevariable region from RNA of antibody-producing cells. See, for example.Larrick, et al., Methods: a Companion to Methods in Enzymology, Vol. 2,page 106 (1991).

The invention contemplates human and humanized forms of non-human (e.g.murine) antibodies. Such humanized antibodies are chimericimmunoglobulins, immunoglobulin chains or fragments thereof (such as Fv,Fab, Fab′, F(ab′ h or other antigen-binding subsequences of antibodies)that contain a minimal sequence derived from non-human immunoglobulin,such as the epitope recognising sequence. For the most part, humanizedantibodies are human immunoglobulins (recipient antibody) in whichresidues from a complementary determining region (CDR) of the recipientare replaced by residues from a CDR of a nonhuman species (donorantibody) such as mouse, rat or rabbit having the desired specificity,affinity and capacity. Humanized antibody(es) containing a minimalsequence(s) of antibody(es) of the invention, such as a sequence(s)recognising the epitope(s) described herein, is one of the preferredembodiments of the invention.

In some instances. Fv framework residues of the human immunoglobulin arereplaced by corresponding non-human residues. Furthermore, humanizedantibodies may comprise residues that are found neither in the recipientantibody nor in the imported CDR or framework sequences. Thesemodifications are made to further refine and optimize antibodyperformance. In general, humanized antibodies will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. For further details, see: Jones et al., 1986, Nature321, 522-525; Reichmann et al., 1988. Nature 332, 323-329; Presta, 1992,Curr Op Struct Biol 2:593-596; Holmes et al., 1997, J Immunol158:2192-2201 and Vaswani, et al., 1998, Annals Allergy, Asthma &Immunol 81:105-115.

The generation of antibodies may be achieved by any standard methods inthe art for producing polyclonal and monoclonal antibodies using naturalor recombinant fragments of human human erythropoietin, thrombopoietin,granulocyte-macrophage colony-stimulating factor (GM-CSF), ciliaryneurotrophic factor (CNFT), interleukin-6 (IL-6), interleukin-3 (IL-3),interferon alpha-1, interferon beta, interferon gamma, cell stem factor,multiple coagulation factor deficiency protein 2 (MCD2) comprising asequence selected from any of the sequences identified as SEQ ID NO:1-41 as an antigen. Such antibodies may be also generated using variantsor fragments of peptide fragments of SEQ ID NOs:1-41, said fragmentsbeing immunogenic fragments which meet at least two of the followingcriteria:

-   (i) being a contiguous amino acid sequence of at least 6 amino    acids;-   (ii) comprising an amino acid sequence derived from the sequence of    human human erythropoietin, thrombopoietin, granulocyte-macrophage    colony-stimulating factor (GM-CSF), ciliary neurotrophic factor    (CNFT), interleukin-6 (IL-6), interleukin-3 (IL-3), interferon    alpha-1, interferon beta, interferon gamma, cell stem factor or    multiple coagulation factor deficiency protein 2 (MCD2).

The antibodies may also be produced in vivo by the individual to betreated, for example, by administering an immunogenic fragment accordingto the invention to said individual. Accordingly, the present inventionfurther relates to a vaccine comprising an immunogenic fragmentdescribed above.

The application also relates to a method for producing an antibody ofthe invention said method comprising a step of providing of animmunogenic fragment described above.

The invention relates both to an antibody, which is capable ofmodulating such as enhancing or attenuating, biological function ofhuman erythropoietin, thrombopoietin, granulocyte-macrophagecolony-stimulating factor (GM-CSF), ciliary neurotrophic factor (CNFT),interleukin-6 (IL-6), interleukin-3 (IL-3), interferon alpha-1,interferon beta, interferon gamma, cell stem factor or multiplecoagulation factor deficiency protein 2 (MCD2), in particular a functionrelated to neural cell growth, differentiation, survival and/orplasticity, and to an antibody, which can recognise and specificallybind the latter proteins without modulating biological activity thereof.

The invention relates to use of the above antibodies for 1) therapeuticapplications when the modulation of activity of human erythropoietin,thrombopoietin, granulocyte-macrophage colony-stimulating factor(GM-CSF), ciliary neurotrophic factor (CNFT), interleukin-6 (IL-6),interleukin-3 (IL-3), interferon alpha-1, interferon beta, interferongamma, cell stem factor or multiple coagulation factor deficiencyprotein 2 (MCD2) is needed, 2) detecting and/or monitoring the latterproteins in vitro and/or in vivo for diagnostic purposes, 3) researchpurposes,

Pharmaceutical Composition

The invention also relates to a pharmaceutical composition comprisingone or more of the compounds defined above, wherein the compound iscapable of stimulating neurite outgrowth and/or neural celldifferentiation, survival of neural cells and/or stimulating learningand/or memory. Thus, the invention concerns a pharmaceutical compositioncapable of stimulating differentiation of neuronal cells and/orstimulating regeneration of neuronal cells, and/or stimulating neuronalplasticity in connection with learning and memory, and/or stimulatingsurvival of neural cells.

In the present context the term “pharmaceutical composition” is usedsynonymously with the term “medicament”.

In a composition the peptide sequences may be formulated as comprisingisolated individual peptide fragments or multimers or dimers thereof asdiscussed above.

The pharmaceutical composition may have the described above effects oncells in vitro or in vivo, wherein the composition is administered to asubject.

The medicament of the invention comprises an effective amount of one ormore of the compounds as defined above, or a composition as definedabove in combination with the pharmaceutically acceptable additives.Such medicament may suitably be formulated for oral, percutaneous,intramuscular, intravenous, intracranial, intrathecal,intracerebroventricular, intranasal or pulmonal administration.

Strategies in formulation development of medicaments and compositionsbased on the compounds of the present invention generally correspond toformulation strategies for any other protein-based drug product.Potential problems and the guidance required to overcome these problemsare dealt with in several textbooks, e.g. “Therapeutic Peptides andProtein Formulation. Processing and Delivery Systems”, Ed. A. K. Banga,Technomic Publishing AG, Basel, 1995.

Injectables are usually prepared either as liquid solutions orsuspensions, solid forms suitable for solution in, or suspension in,liquid prior to injection. The preparation may also be emulsified. Theactive ingredient is often mixed with excipients which arepharmaceutically acceptable and compatible with the active ingredient.Suitable excipients are, for example, water, saline, dextrose, glycerol,ethanol or the like, and combinations thereof. In addition, if desired,the preparation may contain minor amounts of auxiliary substances suchas wetting or emulsifying agents, pH buffering agents, or which enhancethe effectiveness or transportation of the preparation.

Formulations of the compounds of the invention can be prepared bytechniques known to the person skilled in the art. The formulations maycontain pharmaceutically acceptable carriers and excipients includingmicrospheres, liposomes, microcapsules, nanoparticles or the like.

The preparation may suitably be administered by injection, optionally atthe site, where the active ingredient is to exert its effect. Additionalformulations which are suitable for other modes of administrationinclude suppositories, nasal, pulmonal and, in some cases, oralformulations. For suppositories, traditional binders and carriersinclude polyalkylene glycols or triglycerides. Such suppositories may beformed from mixtures containing the active ingredient(s) in the range offrom 0.5% to 10%, preferably 1-2%. Oral formulations include suchnormally employed excipients as, for example, pharmaceutical grades ofmannitol, lactose, starch, magnesium stearate, sodium saccharine,cellulose, magnesium carbonate, and the like. These compositions takethe form of solutions, suspensions, tablets, pills, capsules, sustainedrelease formulations or powders and generally contain 10-95% of theactive ingredient(s), preferably 25-70%.

Other formulations are such suitable for nasal and pulmonaladministration. e.g. inhalators and aerosols.

The active compound may be formulated as neutral or salt forms.Pharmaceutically acceptable salts include acid addition salts (formedwith the free amino groups of the peptide compound) and which are formedwith inorganic acids such as, for example, hydrochloric or phosphoricacids, or such organic acids as acetic acid, oxalic acid, tartaric acid,mandelic acid, and the like. Salts formed with the free carboxyl groupmay also be derived from inorganic bases such as, for example, sodium,potassium, ammonium, calcium, or ferric hydroxides, and such organicbases as isopropylamine, trimethylamine, 2-ethylamino ethanol,histidine, procaine, and the like.

The preparations are administered in a manner compatible with the dosageformulation, and in such amount as will be therapeutically effective.The quantity to be administered depends on the subject to be treated,including, e.g. the weight and age of the subject, the disease to betreated and the stage of disease. Suitable dosage ranges are per kilobody weight normally of the order of several hundred μg activeingredient per administration with a preferred range of from about 0.1μg to 5000 μg per kilo body weight. Using monomeric forms of thecompounds, the suitable dosages are often in the range of from 0.1 μg to5000 μg per kilo body weight, such as in the range of from about 0.1 μgto 3000 μg per kilo body weight, and especially in the range of fromabout 0.1 μg to 1000 μg per kilo body weight. Using multimeric forms ofthe compounds, the suitable dosages are often in the range of from 0.1μg to 1000 μg per kilo body weight, such as in the range of from about0.1 μg to 750 μg per kilo body weight, and especially in the range offrom about 0.1 μg to 500 μg per kilo body weight such as in the range offrom about 0.1 μg to 250 μg per kilo body weight. In particular whenadministering nasally smaller dosages are used than when administeringby other routes. Administration may be performed once or may be followedby subsequent administrations. The dosage will also depend on the routeof administration and will vary with the age and weight of the subjectto be treated. A preferred dosage of multimeric forms would be in theinterval 1 mg to 70 mg per 70 kg body weight.

For some indications a localised or substantially localised applicationis preferred.

Some of the compounds of the present invention are sufficiently active,but for some of the others, the effect will be enhanced if thepreparation further comprises pharmaceutically acceptable additivesand/or carriers. Such additives and carriers will be known in the art.In some cases, it will be advantageous to include a compound, whichpromotes delivery of the active substance to its target.

In many instances, it will be necessary to administrate the formulationmultiple times. Administration may be a continuous infusion, such asintraventricular infusion or administration in more doses such as moretimes a day, daily, more times a week, weekly, etc. it is preferred thatadministration of the medicament is initiated before or shortly afterthe individual has been subjected to the factor(s) that may lead to celldeath. Preferably the medicament is administered within 8 hours from thefactor onset, such as within 5 hours from the factor onset. Many of thecompounds exhibit a long term effect whereby administration of thecompounds may be conducted with long intervals, such as 1 week or 2weeks.

In connection with the use in nerve guides, the administration may becontinuous or in small portions based upon controlled release of theactive compound(s). Furthermore, precursors may be used to control therate of release and/or site of release. Other kinds of implants and wellas oral administration may similarly be based upon controlled releaseand/or the use of precursors.

As discussed above, the present invention relates to treatment ofindividuals for inducing differentiation, stimulating regeneration,plasticity and survival of neural cells in vitro or in vivo, saidtreatment involving administering an effective amount of one or morecompounds as defined above.

Another strategy for administration is to implant or inject cellscapable of expressing and secreting the compound in question. Therebythe compound may be produced at the location where it is going to act.

Treatment

In a further aspect, the present invention relates to said peptides,fragments, or variants thereof for use in the induction ofdifferentiation and/or stimulation of regeneration, plasticity and/orsurvival of neural cells. The use is for the treatment for preventingdiseases and conditions of the central and peripheral nervous system,and of the muscles or of various organs.

Treatment by the use of the compounds/compositions according to theinvention is in one embodiment useful for inducing differentiation,modulating proliferation, stimulate regeneration, neuronal plasticityand survival of cells being implanted or transplanted. This isparticularly useful when using compounds having a long term effect.

Thus, the treatment comprises treatment and/or prophylaxis of cell deathin relation to diseases or conditions of the central and peripheralnervous system, such as postoperative nerve damage, traumatic nervedamage, e.g. resulting from spinal cord injury, impaired myelination ofnerve fibers, postischaemic damage, e.g. resulting from a stroke,multiinfarct dementia, multiple sclerosis, nerve degeneration associatedwith diabetes mellitus, neuro-muscular degeneration, schizophrenia.Alzheimer's disease, Parkinson's disease, or Huntington's disease.

Also, in relation to diseases or conditions of the muscles includingconditions with impaired function of neuro-muscular connections, such asgenetic or traumatic atrophic muscle disorders; or for the treatment ofdiseases or conditions of various organs, such as degenerativeconditions of the gonads, of the pancreas, such as diabetes mellitustype I and II, of the kidney, such as nephrosis the compounds accordingto the invention may be used for inducing differentiation, modulatingproliferation, stimulate regeneration, neuronal plasticity and survival,i.e. stimulating survival.

In yet a further embodiment the use of the compound and/orpharmaceutical composition is for the stimulation of the ability tolearn and/or of the short and/or long term memory.

In particular the compound and/or pharmaceutical composition of theinvention may be used in the treatment of clinical conditions, such aspsychoses, such as senile and presenile organic psychotic conditions,alcoholic psychoses, drug psychoses, transient organic psychoticconditions. Alzheimer's disease, cerebral lipidoses, epilepsy, generalparesis [syphilis], hepatolenticular degeneration, Huntington's chorea,Jakob-Creutzfeldt disease, multiple sclerosis. Pick's disease of thebrain, syphilis, Schizophrenic disorders, affective psychoses, neuroticdisorders, personality disorders, including character neurosis,nonpsychotic personality disorder associated with organic brainsyndromes, paranoid personality disorder, fanatic personality, paranoidpersonality (disorder), paranoid traits, sexual deviations anddisorders, mental retardation, disease in the nerve system and senseorgans, cognitive anomalies, inflammatory disease of the central nervoussystem, such as meningitis, encephalitis, Cerebral degenerations such asAlzheimer's disease, Pick's disease, senile degeneration of brain,communicating hydrocephalus obstructive hydrocephalus, Parkinson'sdisease including other extra pyramidal disease and abnormal movementdisorders, spinocerebellar disease, cerebellar ataxia, Marie's,Sanger-Brown, Dyssynergia cerebellaris myoclonica, primary cerebellardegeneration, such as spinal muscular atrophy, familial, juvenile, adultspinal muscular atrophy, motor neuron disease, amyotrophic lateralsclerosis, motor neuron disease, progressive bulbar palsy, pseudobulbarpalsy, primary lateral sclerosis, other anterior horn cell diseases,anterior horn cell disease, unspecified, other diseases of spinal cord,Syringomyelia and syringobulbia, vascular myelopathies, acute infarctionof spinal cord (embolic) (nonembolic), arterial thrombosis of spinalcord, edema of spinal cord, subacute necrotic myelopathy, subacutecombined degeneration of spinal cord in diseases classified elsewhere,myelopathy, drug-induced, radiation-induced myelitis, disorders of theautonomic nervous system, disorders of peripheral autonomic,sympathetic, parasympathetic, or vegetative system, familialdysautonomia [Riley-Day syndrome], idiopathic peripheral autonomicneuropathy, carotid sinus syncope or syndrome, cervical sympatheticdystrophy or paralysis; peripheral autonomic neuropathy in disordersclassified elsewhere, amyloidosis, diseases of the peripheral nervesystem, brachial plexus lesions, cervical rib syndrome, costoclavicularsyndrome, scalenus anterior syndrome, thoracic outlet syndrome, brachialneuritis or radiculitis, including in newborn, Inflammatory and toxicneuropathy, including acute infective polyneuritis, Guillain-Barresyndrome, Postinfectious polyneuritis, polyneuropathy in collagenvascular disease, disorders affecting multiple structures of eye,purulent endophthalmitis, diseases of the our and mastoid process,abnormality of organs and soft tissues in newborn, including in thenerve system, complications of the administration of anesthetic or othersedation in labor and delivery, diseases in the skin includinginfection, insufficient circulation problem, injuries, including aftersurgery, crushing injury, burns. Injuries to nerves and spinal cord,including division of nerve, lesion in continuity (with or without openwound), traumatic neuroma (with or without open wound), traumatictransient paralysis (with or without open wound), accidental puncture orlaceration during medical procedure, injury to optic nerve and pathways,optic nerve injury, second cranial nerve, injury to optic chasm, injuryto optic pathways, injury to visual cortex, unspecified blindness,injury to other cranial nerve(s), injury to other and unspecifiednerves. Poisoning by drugs, medicinal and biological substances, geneticor traumatic atrophic muscle disorders; or for the treatment of diseasesor conditions of various organs, such as degenerative conditions of thegonads, of the pancreas, such as diabetes mellitus type I and II, of thekidney, such as nephrosis.

A further aspect of the invention is a process of producing apharmaceutical composition, comprising mixing an effective amount of oneor more of the compounds of the invention, or a pharmaceuticalcomposition according to the invention with one or more pharmaceuticallyacceptable additives or carriers, and administer an effective amount ofat least one of said compound, or said pharmaceutical composition to asubject.

In one embodiment of the process as mentioned above, the compounds areused in combination with a prosthetic device, wherein the device is aprosthetic nerve guide, Thus, in a further aspect, the present inventionrelates to a prosthetic nerve guide, characterised in that it comprisesone or more of the compounds or the pharmaceutical composition asdefined above. Nerve guides are known in the art.

Another aspect of the invention relates to the use of a compound asdefined above. In particular the use of a compound according to theinvention is for the production of a pharmaceutical composition. Thepharmaceutical composition is preferably for the treatment orprophylaxis of any of the diseases and conditions mentioned above.

In yet a further aspect the invention relates to a method of treating adisease or condition as discussed above by administering a compound asdefined herein.

EXAMPLES Example 1 Stimulation of Neurite Outgrowth in vitro

Methods:

Cerebellar granule neuron (CGN) cultures were obtained from 3-4-day-oldWistar rats (Charles River, Sulzfeld, Germany, or Moellegaard, Denmark)as previously described by Schousboe et al. (1989). Briefly, thecerebella were dissected, cleared of meninges and blood vessels,chopped, and trypsinized. The neurons were washed in the presence ofDNAse 1 and soybean trypsin inhibitor (Sigma), and cellular debris waspelleted by centrifugation; the cells resuspended and then plated onpoly-L-lysine (PLL; Sigma)-coated or uncoated microtiter plates inNeurobasal medium supplemented with 4% (w/v) bovine serum albumin (BSA),2% (v/v) B27, 1% (v/v) glutamax, 100 U/ml penicillin, 100 gi/mlstreptomycin, 4.5 g D-glucose/L (Sigma) 0.25% (v/v) sodium pyruvate, and2% (v/v) 1 M HEPES (Gibco BRL).

Results and Conclusions

Three out of four Epo fragments (SEQ ID NO: 2, 3, and 4) strongly induceneurite outgrowth from CGN in a dose dependent manner, whereas therecombinant Epo protein (rhEPO) and the peptide having the sequence SEQID NO: 1 do net have any neuritogenic activity. The effect of 24 htreatment of CGN cultures with the peptides and rhEPO is demonstrated inFIGS. 1-5.

Example 2 Stimulation of Survival of Neural Cells in vitro

Methods:

Primary cultures of CGN were plated at a density of 100 000 cells/cm² onpoly-L-lysine coated 8-well permanox slides in Neurobasal-A medium(Gibco BRL) supplemented with 2% (v/v) B27, 0.5% (v/v) glutamax, 100units/mL penicillin, 100 μg/mL streptomycin and KCl, making the finalconcentration of KCl in the medium 40 mm, Twenty-four hours afterplating, Ara-C (Sigma-Aldrich) was added to a final concentration of 10μm to avoid proliferation of glial cells, after which the neurons wereallowed to differentiate for a further 6 days at 37° C. Apoptotic celldeath was induced by washing twice and changing the medium to BasalMedium Eagle (BME; Gibco BRL) supplemented with 1% (v/v) glutamine, 100U/mL, penicillin and 100 μg/mL streptomycin, 3.5 g d-glucose/L and 1%(v/v) sodium pyruvate (Gibco BRL) together with various concentrationsof peptide. Thereby the concentration of potassium in the cultures wasreduced to 5 mm KCl (D'Mello et al. 1993). Two days after induction ofapoptosis, the cells were fixed with 4% (v/v) formaldehyde and stainedwith Hoechst 33258 as described for the survival assay employinghippocampal neurons.

Results and Conclusions:

All Epo-derived peptides and the recombinant Epo protein are capable ofprotection of CGN induced to undergo apoptosis. The effect isdose-dependent. The Epo-derived peptides 2, 3 and 4 (SEQ ID NOs: 2-4)have the strongest neuroprotective effect, which is comparable to theeffect of insulin growth factor-1 (IGF-1), a known strong promoter ofcell survival The effect of the treatment of CGN cultures with thepeptides is demonstrated in FIGS. 6-10.

Example 3 Stimulation of proliferation of haemopoietic cells

Methods:

Evaluation of the effect of four EPO-derived peptides on cellproliferation was performed using a TF-1 cell line. The TF-1 cell linewas established from sample from patient with severe pancytopenia (humanerythroleukemia). The growth of these cells is dependent of the presenceof the granulocyte-macrophage colony-stimulating factor (GM-SCF). TheTF-1 cells were grown in Dulbecco's Modified Eagle Medium (DMEM)supplemented with 10% (v/v) heat inactivated foetal calf serum (FCS), 2mM GlutaMAX, penicillin (100 U/ml) and streptomycin (100 μg/ml), 10 mMHEPES, 1 mM sodium pyruvate, 1% fungizone. The growth medium wassupplemented with 2 ng/ml GM-CSF. Cell proliferation was determinedusing a Biotrak ELISA system, version 2 (Amersham Biosciences, UK). Theprocedure is based on measurement of the levels of5-bromo-2′-deoxyuridine (BrdU) incorporation during DNA synthesis inproliferating cells. Briefly, passaged cells were washed once withstarving medium (culturing medium without GM-CSF) then plated in 96-wellNuclon culture microplates (Nuno, Denmark) at a density of 5000cells/well and grown in starving medium containing 0.008, 0.04, 0.2,1.0, 5.0, 25 and 50 μg/ml of the tested compounds. As a positive controlthe human recombinant erythropoietin (EPO) (Calbiochem, Denmark) wasused in a concentration of 2.0 ng/ml. TF-1 cells were allowed to grow inthe presence of peptides for 24 h and than to all wells (except negativecontrols) BrdU was added to a final concentration of 10 μM and cultureswere further incubated for 24 h, fixed and processed according to theprocedure recommended by manufacturer,

Results/conclusions:

The recombinant Epo protein in a concentration of 2 ng/ml stimulatesproliferation of TF-1 cells. The Epo-derived peptides, Epo3 and Epo4,(SEQ ID NOs:3 and 4) stimulate proliferation of TF-1 cells in aconcentration of 50 μg/ml, but not in concentration 25 μg/ml or below.The Epo-derived peptide 2, Epo2, (SEQ ID NO:2) stimulates proliferationof TF-1 cells in concentrations of 25 and 50 μg/ml, but not inconcentration below 25 μg/ml. The Epo-derived peptide 1. Epo1,stimulates proliferation of TF-1 cells in a concentration of 50 μg/mland probably had a stimulatory effect in a concentration of 0.2 μg/ml.

General conclusion

All four tested Epo peptides Epo1, 2, 3 and 4 (SEQ ID NOs:1-4) possessbiological activity. However, biological activity of Epo fragments andthe full-length recombinant Epo protein is different. Thus, peptidesEpo2, 3 and 4 are capable of stimulating neurite outgrowth, whereas thefull-length recombinant Epo protein does not possess neurite stimulatoryactivity, nor does the Epo1 peptide. However, all peptides and the Epoprotein as well are potent in stimulating neuronal cells, survival. Allpeptides and the Epo protein are also capable of stimulatinghaemopoietic cell proliferation, most potent among them being the Epopeptides 3 and 4. Interestingly, biological activity of different Epopeptides depends on their concentration in cell medium. Thus, theneuritogenic activity of both Epo peptides 2, 3 and 4 is higher when thepeptides are present in low concentrations, whereas stimulation of cellproliferation demands the presence of significantly larger amounts ofthe peptides.

Example 4 Therapeutic Effects of Erythropoietin Mimetic Peptides Epo 2,3 and 4 in Rat Brain in Vivo

Experimental Setup:

Male rats (wistar) (tree groups, 5 rats/group) were injected s.c withdifferent Epo peptides (each group individually with Epo 2, Epo 3 or Epo4) three times according to the scheme:

-   -   1. the day before the lesion: a single injection of a peptide        per animal (1 mg/100 g animal weight);    -   2. the 1^(st) day after the lesion: a single injection of a        peptide per animal (1 mg/100 g animal weight):    -   3. the 2^(nd) day after the lesion: a single injection of a        peptide per animal (1 mg/100 g animal weight);    -   4. the 3^(rd) day after the lesion: animals are sacrificed.

Control groups were a group (4 rats) injected with the vehicle (water)and a group (4 rats), injected with human recombinant EPO (hrEPO) indose 50 μg/100 g rat according to the above scheme.

Methods

1. Traumatic Brain Injury

A traumatic brain injury (TBI) was induced by applying dry ice (−78 C)to the extracranial surface of the skull for 60 sec, which induces afocal injury in the right fronto-parietal cortex. Three days after thelesion (3 dpl) rats were deeply anesthetized with Brietal (10 mg/100 gr)and fixed by transcardial perfusion with 0.9% NaCl and 0.3% heparin,followed by Zamboni's fixative. The brains were removed, dehydratedaccording to the standard protocol, embedded in paraffin, and cut in6-μm-thick frontal sections for further evaluation byimmunohistochemistry and TUNEL.

2. Immunocytochemistry

For immunohistochemistry the sections of the brains were incubatedovernight with polyclonal rabbit anti-cow GFAP, GAP-43, 8-oxoguanine, orPSA-NCAM antibodies 1:250 (Dakopatts). The primary antibodies weredetected in the sections with biotinylated secondary antibodies followedby incubation with streptavidin-biotin-peroxidase (30 min). Afterwards,the sections were incubated with biotinylated tyramide andstreptavidin-peroxidase complex (NEN, Life Science Products, USA, codeNEL700A) prepared following the manufacturer recommendations, andvisualized with 0.015% H₂O₂ in DAB/TBS for 10 min.

3. TUNEL (In Situ Detection of DNA Fragmentation):

Terminal deoxynucleotidyl transferase (TdT)-mediated biotin-linkeddeoxynucleotide nick end labeling (TUNE-1) was performed using theFragment End Labeling (FragEL™) Detection Kit (Calbiochem, USA, codeQIA33). The FragEL kit contains all the materials used below and eachstep was performed according to the manufacturer recommendations. Thetissue was processed and rehydrated as mentioned above, and sectionswere incubated with 20 μg/ml (proteinase K for 20 min to strip offnuclear proteins. After immersion in equilibration buffer for 20 min,sections were incubated with TdT and biotin-labeled deoxynucleotides(dNTP-biotin) in a humified chamber at 37° C. for 1.5 hr. This wasfollowed by washing with the buffer and stop solution for 5 min at roomtemperature to stop the reaction. After washing in TBS and incubation inblocking buffer for 10 min, the sections were incubated withPeroxidase-Streptavidin for 30 min and afterwards, DAB was used aschromogen. The sections were counterstained with methyl-green.

Results:

1. The EPO mimetics clearly increase astrocytosis in the rat brain afterTBI as judged by GFAP IHC. The mast pronounced increase in reactiveastrogliosis is obtained after treating the rats with the Epo 2 peptide;and then with the Epo 3 peptide. The Epo 4 peptide has the least effect.Treatment with the EPO protein results in slightly increasedastrocytosis in comparison to control rats (treated with water), whichdoes increases astrocytosis very slightly, if any.

2. The Epo peptides inhibit programmed cell death in the areassurrounding the necrotic cavity after TBI. All groups showTUNEL-positive apoptotic cells after TBI, but the numbers of dying cellsis the highest in control group (treated with water) compared to therats receiving the EPO protein and Epo 2 peptide. The lowest level ofapoptosis is observed in Epo 3 and Epo 4 treated groups of rats.

3. Immunostaining with a marker of oxidative DNA damage, 8-oxoguanine,demonstrates a higher number of the 8-oxoguanine positive cells in thecontrol group. The EPO and Epo 4 treated rats have a significantlyreduced oxidative DNA damage relative to control, and the lowest numberof the 8-oxoguanine positive cells is observed in Epo 2 and Epo 3treated rats.

4 EPO mimetic peptides stimulate neuroplasticity and neurite outgrowthin vivo after TBI of rat brain as judged by PSA-NCAM and GAP-43immunostaining of rat brain tissue sections. The Epo 3 and Epo 4treatment significantly increases the level of PSA-NCAM staining both inthe area adjusted to the lesion, of Neural Stem Cells (NSC) and in thesubventricular zone (SVZ), whereas only a few cells in the lesion areaof control rats are PSA-NCAM positive, Treatment with Epo 2 and Epo 3also stimulates neurite outgrowth in the area of cortical lesion and inthe choroid plexus, hippocampus, and hypothalamus.

4. The hematocrit (%) significantly increases after EPO treatment(63.7+−1.9 EPO vs 43.7+−3.35 control, p<0.02). All peptide mimetics Epo2, Epo 3 and Epo 4 have no significant effect on the hematocrit (Ep2,Ep3, Ep4 vs control p>0.02). The results are demonstrated in FIG. 15.

Example 5 Binding of the Epo 3 Peptide to Recombinant ErythropoietinReceptor (EPOR-Fc)

The binding of Epo 3 to EPOR-Fc was studied by SPR analysis. The Epo 3peptide was immobilized on a chip and the chip was incubated withsoluble recombinant EPOR-Fc under standard conditions. Three independentexperiments were performed. The results show that EPOR binds to theimmobilized Epo 3 (ionic bonded with the chip) with association ratek_(a)=2.1*10⁴+/−1.4*10⁴ and dissociation ratek_(d)=4.33*10⁻⁵+/−1.62*10⁻⁵. The affinity of the binding of EPOR to Ep3is 4.48 nM. Epo 3 covalently bonded to the chip does not bind to EPOR.The results of binding are shown on FIG. 16.

The invention claimed is:
 1. A method for treating a condition of the central nervous system selected from Alzheimer's disease, epilepsy, and traumatic brain injury, said method comprising administering to a subject in need thereof an effective amount of (i) a peptide consisting of 10 to 20 consecutive amino acids of SEQ ID NO:3; or (ii) a peptide consisting of 10 to 20 amino acids and having more than 80% homology to 10 to 20 consecutive amino acids of SEQ ID NO:3; wherein said peptide is capable of stimulating neurite outgrowth, and/or promoting survival of neural cells, and/or stimulating neural cell proliferation.
 2. The method of claim 1, wherein said condition of the central nervous system is Alzheimer's disease.
 3. The method of claim 1, wherein the subject is human.
 4. The method of claim 1, wherein the peptide (i) consists of 10 to 15 consecutive amino acids of SEQ ID NO:3, or (ii) consists of 10 to 15 amino acids and has more than 80% homology to 10 to 15 consecutive amino acids of SEQ ID NO:3.
 5. The method of claim 1, wherein the peptide consists of from 10 to 15 consecutive amino acids of SEQ ID NO:3.
 6. The method of claim 1, wherein the peptide consists of the amino acid sequence of SEQ ID NO:3.
 7. The method of claim 1, wherein the arginine amino acid residue at position 12 of SEQ ID NO:3 is arginine or conservatively substituted with a histidine or a lysine amino acid residue in the corresponding position within said peptide.
 8. The method of claim 1, wherein said peptide is administered in a dosage of 0.1 μg to 500 μg per kilogram body weight.
 9. A method for treating a condition of the central nervous system selected from Alzheimer's disease, epilepsy, and traumatic brain injury, said method comprising administering to a subject in need thereof an effective amount of a compound consisting of two or more linked peptides linked via one or more linker groups, each peptide either (i) consisting of 10 to 20 consecutive amino acids of SEQ ID NO:3, or (ii) consisting of 10 to 20 amino acids and having more than 80% homology to 10 to 20 consecutive amino acids of SEQ ID NO:3; wherein each peptide is capable of stimulating neurite outgrowth, and/or promoting survival of neural cells, and/or stimulating neural cell proliferation; wherein said linker groups are selected from the group consisting of peptide bonds forming a polymer of the two or more polypeptides, dendrimeric polymers, Ligand Presenting Assembly (LPA)-type polymers, lysine dendrimers, bovine serum albumin (BSA), lipophilic dendrimers, micelle-like carriers, starburst carbon chain polymer conjugates, and ligand presenting assembly polymers based on derivatives of diethylaminomethane, with the proviso that the amino acid sequence of the linked peptides is not identical to the amino acid sequence of naturally occurring erythropoietin.
 10. The method of claim 9, wherein a dimer or a tetramer of said peptides is administered. 